The primary purpose of our research program is to investigate a new class of room temperature ionic liquids, alkylammonium formates (AAFs), as mobile phase alternatives to organic solvents for liquid chromatography (LC) of proteins. Organic solvents such as methanol and acetonitrile cause significant protein unfolding as mobile phase modifiers to control the resolution of proteins separated by reversed phase LC. Alkylammonium formates such as ethyl-, n- propyl-, and n-butyl-ammonium formates have been synthesized and characterized with respect to viscosity, polarity, UV absorbance, and solvent strength for LC. These ionic liquids can act as replacement solvents for methanol and aceonitrile for the LC separation of pharmaceuticals with UV or fluorescence detection. Fluorescence studies of proteins in these ionic liquids, particularly ethylammonium formate (EAF), have shown less unfolding and exposure of hydrophobic aromatic amino acids such as tryptophan as compared to methanol and acetonitrile. We hypothesize that the short chain AAFs such as methylammonium formate (MAF) and EAF will show the best compatibility with proteins. The synthesis and purification of MAF and EAF will be studied to reduce the UV background down to 220 nm so circular dichroism can be used to study the tertiary and secondary structures of model proteins such as cytochrome c, bovine serum albumin, and lysozyme in these ionic liquids. The activity of enzymes such as hydroxysteroid dehydrogenase should be more stable in mixtures of buffer and MAF or EAF than corresponding buffer-organic solvent solutions permitting their use as analytical reagents. Both reversed phase LC and hydrophobic interaction chromatography (HIC) of proteins will be characterized using MAF and EAF as mobile phase modifiers and improved resolution particularly for HIC is expected. Based on circular dichroism spectroscopy of collected fractions, it is also expected that the native protein conformations will be maintained. The relevance of this proposal to public health is that a new class of ionic liquids, alkylammonium formates, have been found to be more compatible than standard organic solvents for the purification of proteins. The native conformation of the protein is maintained during the chromatographic separation process required for purification. This will benefit the pharmaceutical industry and biotech companies as well as academic or government research programs with an interest in proteins and enzymes as drugs or reagents to facilitate medical research.